PCR et culture du virus dans différents milieux à différents temps :
– Culture crachats se négative avant négativité PCR (cependant la charge virale diminue alors en-dessous d’un certain seuil)
– culture toujours negative dans les selles malgré des PCR fortement positifs et la preuve d’une réplication active : du fait de la forme non sévère de la maladie ou neutralisation du virus dans le tube digestif ?
Conseil pratique pour désengorger les hôpitaux : sortie à domicile après le dixième jour avec un PCR en-dessous d’un certain seuil.
To understand infectivity, live virus isolation was attempted on multiple occasions from clinical samples (Figure 1D). Whereas virus was readily isolatedً during the first week of symptoms from a considerable fraction of samples (16.66% in swabs, 83.33% in sputum samples), no isolates were obtained from samples taken after day 8 in spite of ongoing high viral loads . Virus isolation from stool samples was never successful, irrespective of viral RNA concentration, based on a total of 13 samples taken between days six to twelve from four patients. Virus isolation success also depended on viral load: samples containing < 10 [puissance 6] copies/mL (or copies per sample) never yielded an isolate.
For swab and sputum, interpolation based on a probit model was done to obtain laboratory-based infectivity criteria for discharge of patients (Figures 1E, F). High viral loads and successful isolation from early throat swabs suggested potential virus replication in upper respiratory tract tissues.
To obtain proof of active virus replication in absence of histopathology, we conducted RT-PCR tests to identify viral subgenomic messenger RNAs (sgRNA) directly in clinical samples (extended data Figure S1). Viral sgRNA is only transcribed in infected cells and is not packaged into virions, therefore indicating the presence of actively-infected cells in samples. Viral sgRNA was compared against viral genomic RNA in the same sample. In sputum samples taken on days 4/5, 6/7, and 8/9, a time in which active replication in sputum was obvious in all patients as per longitudinal viral load courses (see below), mean normalized sgRNA per genome ratios were ~0.4% (Figure 1G). A decline occurred over days 10/11. In throat swabs, samples taken up to day 5 were in the same range, while no sgRNA was detectable in swabs thereafter. Together, these data indicate active replication of SARS-CoV-2 in the throat during the first 5 days after symptoms onset. No, or only minimal, indication of replication in stool was obtained by the same method (Figure 1G).
The combination of very high virus RNA concentrations and occasional detection of sgRNA-containing cells in stool indicate active replication in the gastrointestinal tract. Our failure to isolate live #SARS-CoV-2 from stool may be due to the mild courses of cases, with only one case showing intermittent diarrhea. Further studies should therefore address whether SARS-CoV-2 shed in stool is rendered non-infectious though contact with the gut environment.
Our initial results suggest that measures to contain viral spread should aim at droplet-, rather than fomite-based transmission.
The prolonged viral shedding in sputum is relevant not only for hospital infection control, but also for discharge management. In a situation characterized by limited capacity of hospital beds in infectious diseases wards, there is pressure for early discharge following treatment. Based on the present findings, early discharge with ensuing home isolation could be chosen for patients who are beyond day 10 of symptoms with less than 100,000 viral RNA copies per ml of sputum. Both criteria predict that there is little residual risk of infectivity, based on cell culture.